1. Field of the Invention
This invention relates to the recognition and control of bacterial infections, particularly infections caused by Staphylococcus aureus (S. aureus). More particularly, the present invention provides a staphylococcal accessory regulatory protein sarA, and the gene encoding that protein (sar).
2. Description of the Related Art
Septicemia due to Staphylococcus aureus is often the consequence of a local infection that has gained access to the bloodstream. Once the bacteria enter the bloodstream, patients are at a risk of developing a range of infections from endocarditis, pneumonia, meningitis, and sepsis to severe toxemia (including toxic shock syndrome) and other life-threatening metastatic complications. Although the use of newer antimicrobial agents has initially controlled some of these infections, the recent emergence of multiple-drug resistance in S. aureus has made many currently available antimicrobial agents (including fluoroquinones) ineffective, thereby posing an important public health problem. Thus, there is an urgent need for alternative approaches in the treatment of S. aureus infections.
Infections caused by S. aureus are probably related to the organism's striking capability to react to changing environments during the infection process. In any stage of surface colonization, entry, and invasion, this highly coordinated response seems to be modulated by the expression of appropriate genes via signal transduction pathways.
One potentially effective mechanism for dealing with the emergence of antibiotic resistance among S. aureus strains is the development of safe and effective vaccines. However, experimental data justifying a direct vaccine strategy are presently lacking. An alternative approach is to consider targeting regulatory loci that are involved in the control and expression of potential virulence determinants (e.g., hemolysins and fibrinogen and fibronectin binding proteins).
Postexponential phase expression in bacteria is generally governed by global regulatory systems in which a common regulator controls the activities of several unlinked genes. Many exoproteins normally synthesized and secreted during the postexponential phase in S. aureus are virulence factors. In contrast, synthesis of a number of surface proteins (e.g., protein A, coagulase, and fibronectin binding proteins) that clearly play a role in staphylococcal infections is repressed postexponentially. The regulation of virulence determinants and other exoprotein genes in S. aureus involves at least three global regulatory systems, including agr, xpr, and sar.
The agr locus controls production of extracellular and cell wall proteins that appear to play a role in virulence (e.g., hemolysins, coagulase, and protein A). Most of the exoprotein regulated by agr are either not synthesized or synthesized at a reduced rate in agr mutants while the synthesis of surface proteins is upregulated (11). The agr locus has been cloned (id.) and consists of at least five genes, agrA, agrB, agrC, agrD and the hld (.beta.-hemolysin) gene. Sequence analysis indicated that it has features suggestive of a two component regulatory system as described in other procaryotes (11). In particular, the agrB is the signaling component while agrA corresponds to the transcription activation element (11, 14). The agr locus is composed of two divergent transcription units designated RNAII (agr A,B,C and D genes) and RNAIII (hld gene). Mutations in either agrA or agrB has led to decreased transcription of RNAIII (11, 16), which also encodes the 26 residue hemolysin polypeptide, is essential for the transcriptional control of exoprotein synthesis (e.g. .alpha.-hemolysin) (11).
The xpr locus, like that of agr, also positively regulates exoprotein synthesis. Smeltzer et al. reported that two sets of agr and xpr mutants with different genetic backgrounds were less lethal and their corresponding parents in a mouse peritonitis model (Smeltzer et al. 1993. Infect. Immun. 61:919-925); however, a very high challenging inoculum (10.sup.9 -10.sup.10 CFU) was required for lethality in this study. Furthermore, both xpr and agr mutants produced greatly reduced amount of hemolysin. This finding together with the observation that the RNAIII level is decreased in a xpr mutant suggest that the xpr and agr loci may behave as interactive regulatory genes (9,20).
An additional locus in S. aureus, designated sar, that is involved in the regulation of exoproteins has been reported (3). Inactivation of this locus by Tn917LTV1 insertion has resulted in decreased expression of several extracellular (e.g. .beta.-hemolysin) and cell wall proteins (3). Phenotypic, Southern blot and genetic mapping analyses indicated that this locus is distinct from agr and xpr (3,4). Using the DNA sequence flanking the Tn917LTV1 insertion as a probe, the sar gene that is involved in the regulation of exoprotein synthesis has been cloned and sequenced. Additional transcriptional and phenotypic studies revealed that this sar gene is necessary for the optimal expression of agr.
Inactivation of the sar locus has resulted in alterations of expression of exoproteins in three different S. aureus isolates (strains DB, RN6390 and RN450) (3,4). Using both .alpha. and .beta. hemolysin genes as probes, transcriptional studies of strains with well-defined genetic backgrounds (i.e. RN6390 and RN450) revealed that the sar locus probably regulates exoprotein genes positively at the MRNA level (4). The regulation of exoprotein genes (e.g..alpha. and .beta.-hemolysins) by the sar locus in vitro was found to begin at midlog phase and continued onto the postexponential phase (4). This mode of regulation is similar to that of agr on target exoprotein gene transcription.
To elucidate the interaction between the sar and agr loci, the level of the RNAIII transcript in sar mutants as well as mutants complemented was assayed with an intact sarA gene. The data suggested that the levels of RNAIII were related to a functional sarA gene (FIG. 2). It was also found possible to overcome the deficiency in .beta. and .alpha. hemolysin expression in a sar mutant by introducing a plasmid carrying RNAIII under the control of a promoter uninfluenced by sar. To rule out the possibility of some concerted interaction, Northern blot analysis was employed to determine that the level of sar mRNA did not appear to be altered appreciably in an agr background (RN6911) (11) as compared to the wild type parent RN6390. Taken together, these data suggest that the agr locus is under the control of sar.
Analysis of the sarA gene sequence leads to several interesting observations. First, there is no helix-turn-helix motif identifiable in a protein sequence that has a predicted cL helical conformation. Secondly, glycine residues which are frequently found in helix-turn-helix motif (1) as well as in two component signal transduction systems (14) are noticeably absent. Third, a small molecular size together with a high percentage of charged residues (33%) and a basic charge are molecular properties that are consistent with those found in other DNA binding proteins (21). Fourth, in contrast to the agr locus, direct sequence comparison of the sarA gene with prototypic sensor and activator genes in E. coli, S. typhimurium and B. subtilis did not reveal any significant similarity to two component regulatory systems (14). Finally, sequence similarity with virF, which is a positive regulator of invasive genes in a regulon carried on a large plasmid in Shigella flexneri (8,18), is of comparative interest. Like that of virF which regulates target genes via the control of another positive regulatory gene virB, the sarA gene may govern the expression of exoprotein genes (e.g. .alpha. and .beta. hemolysins) by positively controlling the level of RNAIII. However, the exact mechanism by which the sarA gene product interacts with the agr locus is not apparent from the sequence analysis.
The postexponential regulation of exoprotein genes in S. aureus involves at least three global regulatory systems (sar, agr and xpr). Although the evidence suggests that the sarA gene may control exoprotein synthesis via the control of agr, the relationship between sarA and xpr is not clear. Nevertheless, the observation that both agr and xpr mutants produce greatly reduced amounts of RNAIII transcript has led to the idea that agr and xpr loci may behave as interactive regulatory genes. It is therefore, conceivable that the sarA may interact with the xpr locus as well.
It should be noted, however, that the restoration of RNAIII transcript (FIG. 8) upon the introduction of the sarA gene into a sar mutant was never complete. This finding raised the possibility that additional signals may be required for a normal pattern of RNAIII transcription, thereby leading to optimal expression of exoproteins at postexponential phase. Based on the pattern of transcription of an exoprotein gene such as hemolysin in an agr.sup.+ parent, Vandenesch et al suggested that a separate postexponential signal independent of agr may be needed for augmented .alpha.-hemolysin transcription during the postexponential phase (22).